Blood substitute suppression by peroxides

ABSTRACT

The invention concerns a method for the elimination or/and reduction of interferences which are caused by the presence of free haemoglobin in the determination of an analyte in a sample by optical measurement, wherein one or several peroxidic compounds are added to the analytical reagent or to a part thereof before the measurement. In addition a reagent kit is disclosed which contains at least one peroxidic compound.

The invention concerns a method for the determination of an analyte in asample containing free haemoglobin by optical measurement with theaddition of a bleaching agent to the analytical reagent. This method isparticularly suitable for the determination of components of a medicalsample such as the parameters α-amylase, alkaline phosphatase andγ-glutamyl transferase in a blood, serum or plasma sample.

A frequent occurrence when determining components of clinical-diagnosticrelevance in blood, serum and plasma samples is that these samplematerials contain free haemoglobin i.e. they are haemolytic. Thishaemolysis can either be due to native haemoglobin released from theerythrocytes or the therapeutic administration of blood substitutesbased on non-cellular haemoglobin derivatives.

In many cases the analysis of such samples containing haemoglobin isdisturbed or made completely impossible, especially when usingphotometric methods of determination, mainly due to the spectralproperties of haemoglobin or haemoglobin derivatives. This is especiallythe case when the photometric measurement is carried out at a wavelengthat which a strong absorption of haemoglobin takes place for example atwavelengths between ca. 400-420 nm and if at the same time a highcontent of haemoglobin is present in the sample i.e. usually>500 mg/dl.

The availability of blood substitutes based on haemoglobin derivativesposes the problem of eliminating such analytical interference caused byhaemolysis to a much greater extent than previously. After theirtherapeutic administration, haemoglobin levels of up to ca. 2000 mg/dlcan occur in the blood serum or blood plasma and the situation isaggravated by the fact that in this case it is not possible to avoid thepresence of free haemoglobin in the sample even by suitableprecautionary measures when serum or plasma is obtained from the bloodsample.

There is therefore a need for a method which enables photometricdeterminations of diagnostically important analytes to be carried outwithout interference even in strongly haemolytic serum or plasma samplescontaining for example blood substitutes up to concentrations of atleast 2000 mg/dl.

The object of the invention was achieved by a method for eliminatingor/and reducing interferences that are caused by the presence of freehaemoglobin in the determination of an analyte in a sample by opticalmeasurement in which one or several peroxidic compounds are added to thereagent used to determine the analyte or to a part thereof.

The addition according to the invention of peroxidic compounds to theanalytical reagent or to one or several partial reagents enables on theone hand an elimination of spectral interference by the haemoglobinpresent in the sample. On the other hand it was also possible toeliminate interference caused by interactions of haemoglobin with othersubstances present in the sample.

The reagent which is added to the sample can be in a liquid or solidform. In the case of analyte determinations which are carried out in aliquid phase, it is also preferable to add a liquid reagent to thesample. However, in dry tests the reagent can also be present in a solidform e.g. in the form of impregnated fibres or fleeces.

Inorganic as well as organic peroxides come into consideration as theperoxidic substances. Inorganic peroxidic compounds are preferred suchas H₂ O₂, peroxides, perborates, persulfates, peroxodisulfates,percarbonates etc. The peroxidic compounds are particularly preferablyselected from the group comprising H₂ O₂ and perborates such as NaBO₂×H₂ O₂ ×3H₂ O or Na₂ B₄ O₇ ×H₂ O ₂ ×9H₂ O.

The final concentration of the peroxidic compounds in the test mixturecan be varied over a wide range. It is preferably 1-500 mmol/l andparticularly preferably 5 -200 mmol/l with reference to the content ofO₂ ²⁻ in the test mixture.

The addition of peroxidic compounds leads to a rapid bleaching of thecolour caused by the haemoglobin or haemoglobin derivative after mixingthe sample with the analytical reagent without at the same timesignificantly impairing a subsequent determination in the same reactionmixture of the analyte such as e.g. an enzyme by means of chromogenicsubstrates. This finding is very surprising since in no way could ithave been foreseen that peroxidic compounds do not influence enzymaticactivities and/or interfere with the respective indicator reaction evenat relatively high concentrations and within the usual temperature rangeused for photometric serum and plasma analyses i.e. in general from 25to 37° C., even over a relatively long duration of action i.e.preferably for at least 1 min and particularly preferably for at least2-10 min.

The method according to the invention is especially suitable for opticalmeasurements which are carried out at at least one measurementwavelength at which native haemoglobin or synthetic haemoglobinderivatives have an absorption. The method is particularly preferablycarried out with optical measurements in the measurement wavelengthranges of about 380-450 nm and in particular of 400-420 nm or/and520-590 nm where haemoglobin has its main and secondary absorptions.

The method according to the invention is suitable for the determinationof any samples in which free haemoglobin is present. Examples of suchsamples are native haemolytic blood, serum or plasma samples or sampleswhich contain a blood substitute based on haemoglobin derivatives.Examples of blood substitutes which are covered in the sense of thepresent invention by the term "free haemoglobin" are modified orintramolecularly or intermolecularly cross-linked or polymerizedderivatives of haemoglobins especially of human haemoglobin or bovinehaemoglobin e.g. DCL-haemoglobin (diaspirin cross-linked haemoglobin) aswell as recombinant haemoglobin muteins obtained for example frommicroorganisms (cf. e.g. Blood substitutes, R. M. Winslow, K. D.Vandegriff, M. Intaglietta (editor), Birkhauser, Boston 1995 andEP-A-0700 997). In a particular embodiment of the method according tothe invention the analyte to be determined is an enzyme. Preferredenzymes are selected from the group of hydrolases such as e.g.α-amylase, alkaline phosphatase and γ-glutamyl transferase (γ-GT). Inaddition the method according to the invention is also suitable for thedetermination of other analytes.

A medical sample e.g. a blood, serum or plasma sample is preferably usedas the sample in the method according to the invention and in particulara human serum or plasma sample.

A particular advantage of the method according to the invention is thatit can be carried out in an automated analyser e.g. a BoehringerMannheim/Hitachi 704 or 717 analyser.

In a preferred embodiment, the analyte is determined in the methodaccording to the invention as a multi-step test e.g. as a two-step testin which at least two partial reagents are added successively atdifferent times to the sample. In such a multi-step test procedure, theperoxidic compounds can be preferably added to the partial reagent whichis added first to the sample. The actual analyte determination is thenpreferably carried out at the earliest after addition of the secondpartial reagent which for example in the case of an enzyme determinationcan contain a chromogenic colour substrate.

If in a multi-step test procedure the peroxidic compounds are addedtogether with a first partial reagent, the second or at least one of theother partial reagents can then optionally additionally contain an agentwhich in turn removes the excess peroxidic compounds derived from thefirst partial reagent in order to avoid possible interference of otheranalyses that are subsequently carried out in the same reaction vesseldue to so-called carry-over effects.

If H₂ O₂ or/and perborates are used as the peroxides, the agent forremoving the peroxidic compounds preferably contains aperoxide-converting enzyme such as catalase or/and peroxidase optionallytogether with one or several suitable substrates for theperoxide-converting enzyme.

In addition superoxide dismutase can be additionally added to theanalytical reagent or a partial reagent thereof to prevent formation ofsuperoxide radicals O₂ -.

By means of the method according to the invention it is possible toachieve without further measures a recovery of the analyte to bedetected even in haemolytic samples of 100±10% and preferably 100±5% ifat the time of the first measurement the bleaching of haemoglobin or thehaemoglobin derivative by the peroxidic compounds is essentiallycompleted or has come to a standstill. If, in contrast, the bleachingreaction has not yet come to a standstill at the start of themeasurement which may be the case with a very high haemoglobinconcentration in the sample and/or with a relatively high sample toreagent volume ratio, then preferably an additional blank valuecorrection can be carried out to achieve the desired analyticalaccuracy. This blank value correction can for example be carried out bysubtracting the blank signal obtained in the same measurement period ina parallel test with a like-wise peroxide-containing but chromogen-freereagent, from the measurement signal which is obtained with the reagentcontaining peroxide and chromogen. The blank value correction isparticularly preferably carried out by subtracting a kinetic blankvalue.

In a two-step test the blank value is preferably determined by adding afirst partial reagent (R1) containing peroxide, preferably a reagentwith the same peroxide concentration as in the test mixture, andsubsequently a chromogen-free second partial reagent (R2) to the sampleand determining the blank value from this test mixture which is then tobe subtracted from the measured signal.

A further subject matter of the invention is a reagent kit for thedetermination of an analyte in a sample by optical measurement which, inaddition to the components required to determine the analyte, containsat least one peroxidic compound to eliminate or/and reduce interferencescaused by free haemoglobin. The reagent kit is preferably composed of atleast two partial reagents that are spatially separated from oneanother. A partial reagent preferably contains the peroxidic compoundseparate from all other components. In this partial reagent theperoxidic compound can be present in a solid or liquid form e.g. as apowder or tablet or also as a stabilized solution. The partial reagentcontaining the peroxidic compound is preferably mixed with a furtherpartial reagent before carrying out the test.

A reagent kit according to the invention for the determination ofenzymes preferably contains a first partial reagent containing theperoxidic compound, a further partial reagent that can be mixed with theperoxidic compound and a separate reagent which contains a chromogeniccolour substrate.

A reagent kit according to the invention for the determination ofα-amylase preferably comprises a first partial reagent which containsthe peroxidic compound, a further partial reagent which is compatiblewith the peroxidic compound which optionally contains an α-amylaseauxiliary enzyme such as α-glucosidase or/and an antibody, as well as afurther partial reagent which contains a chromogenic α-amylase substratesuch as an oligomeric glucoside.

A reagent kit according to the invention for the determination ofalkaline phosphatase preferably comprises a first partial reagentcontaining a peroxidic compound. A further partial reagent which iscompatible with the first partial reagent preferably contains a suitablealkaline buffer, and a further partial reagent contains a chromogenicsubstrate for alkaline phosphatase e.g. a phosphate ester such as4-nitrophenyl phosphate.

A reagent kit according to the invention for the determination ofγ-glutamyl transferase preferably comprises a first partial reagentcontaining a peroxidic compound, a second partial reagent which containsa suitable buffer and a further partial reagent which contains achromogenic substrate for γ-GT such asL-γ-glutamyl-3-carboxy-4-nitroanilide.

It is intended to further elucidate the present invention by theattached figures as well as by the following examples.

FIG. 1 shows the time course of the measured signal in a determinationof alkaline phosphatase in samples containing different amounts ofhaemoglobin derivative without the addition of a peroxidic compound;

FIG. 2 shows the time course of the measured signal in a determinationof alkaline phosphatase in samples containing different amounts ofhaemoglobin derivative with the addition of a peroxidic compound (1-4)or the time course of the measured signal for a blank correction(1'-4');

FIG. 3 shows the time course of the measured signal in a determinationof α-amylase in samples containing different amounts of haemoglobinderivative without the addition of a peroxidic compound;

FIG. 4 shows the time course of the measured signal in a determinationof a-amylase in samples containing different amounts of haemoglobinderivative with the addition of a peroxidic compound (1-4) or the timecourse of the measured signal for a blank correction (1'-4');

FIG. 5 shows the time course of the measured signal in a determinationof γ-GT in samples containing different amounts of haemoglobinderivative without the addition of a peroxidic compound;

FIG. 6 shows the time course of the measured signal in a determinationof γ-GT in samples containing different amounts of a HB derivative withthe addition of a peroxidic compound (1-7) and

FIG. 7 shows the time course of the signal for a blank correction(1'-7') carried out in parallel to the experiments in FIG. 6.

EXAMPLES Example 1

Determination of alkaline phosphatase (AP) in serum containing ahaemoglobin derivative.

1.1. Sample Material

0.40 ml aliquots of a human serum pool are supplemented with 0.0-0.10 mlhaemoglobin derivative (di-aspirin cross-linked haemoglobin, `DClHb`, 10g/dl; Baxter Co.) in steps of 0.01 ml and in each case filled up to 0.50ml with 0.9% NaCl solution to equalize the volume (DClHb concentration200-2000 mg/dl).

A mixture of 0.40 ml of the same serum pool with 0.10 ml 0.9% aqueousNaCl solution is also run as a control.

1.2. Reagents:

1.2.1. Alkaline phosphatase basic reagent (corresponding to therecommendations of the German Society for Clinical Chemistry)

AP DGKCh (Sys 2 pack, Boehringer Mannheim GmbH, order No. 1 662 902),composition:

    ______________________________________                                        R1:    D(-)-N-methylglucamine HCl (pH 10.1)                                                                 610 mmol/l                                                   Mg acetate                                0.61 mmol/l                                                     NaCl                                                                       85.4 mmol/l                               R2: 4-nitrophenylphosphate                            122 mmol/l            ______________________________________                                    

1.2.2. Alkaline phosphatase reagent with peroxide additive:

Like 1.2.1. except that H₂ O₂ is additionally added to R1 at aconcentration of 147 mmol/l (0.5 ml 30% perhydrol per 30 ml R1) (H₂ O₂final concentration in the test=120 mmol/l.

1.3. Procedure for the determination

Alkaline phosphatase was determined on a Boehringer Mannheim/Hitachi 717analyser at 37° C. with the instrument settings according to the workinginstructions of the package insert (5 μl sample, 250 μl R1, 50 μl R2;wavelengths 700/405 nm; measurement interval 30th-38th measurementpoint).

1.4. Results

1.4.1. Reaction kinetics (absorbance time course):

FIG. 1 shows the absorbance time course in the alkaline phosphatase testwhen using the basic reagent (1.2.1) and the following samples:

curve 1: 0.9% NaCl (=reagent blank)

curve 2: serum without DClHb

curve 3: serum containing 1000 mg/dl DClHb

curve 4: serum containing 2000 mg/dl DClHb

At least with the serum with a DClHb content of 2000 mg/dl an initialabsorbance of 2.3 is reached which is at the photometric detection limitand no longer allows a reliable determination of the enzyme activity.

In contrast, as shown in FIG. 2 (assignment of the absorbance timecourses to the various samples as in FIG. 1) the presence of peroxide inR1 of the alkaline phosphatase reagent leads to a rapid decrease of theinitial absorbance caused by the haemoglobin derivative so that aninitial absorbance of less than 0.2 is reached at the start of themeasurement interval (measuring point 30) even with the serum spikedwith DClHb to a concentration of 2000 mg/dl. A further haemoglobinbleaching which may still underly the colour formation from thechromogenic substrate during the measurement interval can be determinedby additionally measuring a separate reaction mixture withchromogen-free R2 (=R1 from the basic reagent) and using this to correctthe AP activity (curves 1'-4' of FIG. 2).

1.4.2. Alkaline phosphatase recovery

The following table lists the data for the recovery of alkalinephosphatase in the serum pool spiked with various DClHb amounts whenusing (a) the conventional AP reagent and (b) the AP reagent to which H₂O₂ was added to R1:

    ______________________________________                                                  Recovery of alkaline phosphatase*                                                       Reagent with H.sub.2 O.sub.2 addition                                                 without blank                                                                          with blank                                  DClHb con- Reagent without value value                                       Sample centration H.sub.2 O.sub.2 addition correction correction            No.   (mg/dl)   U/l     %     U/l   %    U/l  %                               ______________________________________                                        1     0         275     (100) 272   99   272  99                                2    200       254       92             272     99      273     99                                                         3    400       239                                                           87             268     98                                                        270     98                     4    600       232       84             265     96      269     98                                                         5    800       216                                                           78             262     95                                                        267     97                     6    1,000     200       73             262     95      268     98                                                         7    1,200     188                                                           68             259     94                                                        267     97                     8    1,400     176       64             262     95      272     99                                                         9    1,600     162                                                           59             258     94                                                        268     98                     10   1,800     146       53             257     94      271     99                                                         11   2,000     139                                                           50             249     91                                                        262     95                   ______________________________________                                         *In each case the tests were calibrated with a calibrator for automated       systems, Boehringer Mannheim GmbH, Order No. 759 350.                    

The table shows that the recovery of alkaline phosphatase using theconventional analytical reagent decreases considerably with increasingsample concentrations of haemoglobin derivative, whereas with theperoxide addition according to the invention it is still over 90%(calculated without a blank value correction) or ≧95% (calculated withblank value correction) even at 2000 mg/dl DClHb/dl.

Identical results are also obtained if the serum pool is spiked with thehaemoglobin derivative from the Somatogen Co. (recombinant humanhaemoglobin derivative from E. coli with an intramolecular di-α fusion)instead of with DClHb.

The result also does not change if sodium perborate (NaBO₂.H₂ O₂.3 H₂ O)is added instead of H₂ O₂ as the peroxide; e.g. at perborateconcentrations of 14.4 or 28.8 mmol/l R1 (final concentration in thetest=11.8 or 23.6 mmol/l) the following AP recoveries were measured:

    ______________________________________                                                     Recovery of alkaline phosphatase (%)                                                          R1 with R1 with                                      DClHb content   R1 without       perborate     perborate,                   Sample  (mg/dl)        perborate     14.4 mmol/l   28.8 mmol/l              ______________________________________                                        1      0           (100)     103*    103*                                       2        2000         67           101*            102*                     ______________________________________                                         *) corrected by subtracting the kinetic blank value (test mixture with        chromogenfree R2)                                                        

Finally similar results were obtained when for example catalase wasadditionally added to R2 to degrade the peroxide.

EXAMPLE 2

Determination of α-amylase in serum containing a haemoglobin derivative.

2.1. Sample material

Type and preparation of example 1, item 1.1.

2.2. Reagents

2.2.1. Amylase basic reagent:

α-amylase EPS liquid (Sys 2 pack, Boehringer Mannheim GmbH, order No.1555 693), composition:

    ______________________________________                                        R1:      α-glucosidase                                                                            > 4 U/ml                                                   HEPES BUFFER (pH 7.15)            52.5 mmol/l                                 NaCl                              87.0 mmol/l                                 MgCl.sub.2                         12.6 mmol/l                           R2:  4,6-ethylidene-G7 PNP             22.0 mmol/l                                 HEPES buffer (pH 7.15)            52.5 mmol/l                          ______________________________________                                    

2.2.2. Amylase reagent with peroxide additive:

Like 2.2.1. except that H₂ O₂ is additionally added to R1 at aconcentration of 147 mmol/l (0.5 ml 30% perhydrol per 30 ml R1) (H₂ O₂final concentration in the test=118 mmol/l).

2.3. Procedure for the determination

Amylase was determined on a Boehringer Mannheim/Hitachi 717 analyser at37° C. with the instrument settings according to the workinginstructions of the package insert (10 μl sample, 250 μl R₁, 50 μl R2;wavelengths 700/405 nm; measurement interval 40th-50th measurementpoint).

2.4. Results

2.4.1. Reaction kinetics (absorbance time course):

FIG. 3 shows the absorbance time course in the amylase test when usingthe basic reagent (2.2.1) and the following samples:

curve 1: 0.9% NaCl (=reagent blank value)

curve 2: serum without DClHb

curve 3: serum containing 1000 mg/dl DClHb

curve 4: serum containing 2000 mg/dl DClHb

FIG. 3 shows that a base absorbance of more than 2 is reached in theconventional amylase test even at a DClHb concentration of 1000 mg/dlwhereas the measuring range of the photometer is exceeded with thesample spiked with DClHb to 2000 mg/dl and thus a determination ofamylase is completely impossible.

In contrast, as shown in FIG. 4 (assignment of the various absorbancetime courses to the sample material as in FIG. 3) the addition ofperoxide to R1 of the amylase reagent leads to a rapid decrease of theinitial absorbance caused by the haemoglobin derivative so that aninitial absorbance of less than ca. 0.3 is reached at the start of themeasurement interval (measuring point 40) even with the serum spikedwith DClHb to 2000 mg/dl. Like the AP determination it is also advisablein this case to also measure a separate reaction mixture withchromogen-free R2 (=R1 from the basic reagent) and using this as acorrection when calculating the amylase activity (curves 1'-4' of FIG.4) in order to take a further haemoglobin bleaching in the measurementwindow into account.

2.4.2. Amylase recovery

The following table lists the data for the recovery of amylase in theserum pool spiked with various DClHb amounts when using (a) theconventional amylase reagent and (b) the amylase reagent to which H₂ O₂was added to R1:

    ______________________________________                                                    Recovery of amylase*                                                      DClHb con-                                                                              Reagent without                                                                           Reagent with                                      Sample   centration   H.sub.2 O.sub.2 addition  H.sub.2 O.sub.2                                           addition**                                      No.     (mg/dl)   U/l    %      U/l    %                                      ______________________________________                                         1      0         122    (100)  119    98                                       2         200         95     78                     114       93                                                    3         400         79     65                                                                120       98                                                 4         600         84     69                                                                117       96                                                 5         800         76     62                                                                115       94                                                 6        1000         83     68                                                                113       93                                                 7        1200         88     72                                                                117       95                                                 8        1400         86     71                                                                114       93                                                 9        1600         69     57                                                                112       92                                                 10       1800         65     53                                                                111       91                                                 11       2000     -13 -11                                                                112       92               ______________________________________                                         *) In each case the tests were calibrated with a calibrator for automated     systems, Boehringer Mannheim GmbH, Order No. 759 350                          **) corrected by subtracting the kinetic blank value (test mixture with       chromogenfree R2).                                                       

The table shows that the recovery of amylase with the conventionalreagent decreases considerably with increasing DClHb concentrations andnegative values are even obtained at 2000 mg/dl, whereas with theperoxide addition according to the invention it is still over 90%.

Otherwise the comments made for the AP determination also apply.

EXAMPLE 3

Determination of γ-glutamyl transferase (γ-GT) in serum containinghaemoglobin derivative

3.1. Sample Material

Type and preparation of example 1, item 1.1.

3.2. Reagents

3.2.1. γ-GT basic reagent:

Gamma-GT (Sys 2 pack, Boehringer Mannheim GmbH, order No. 489 496),composition:

    ______________________________________                                        R1:    NaOH                   76 mmol/l                                             glycylglycine                           150 mmol/l                            (pH 7.7)                                                                  R2: L-γ-glutamyl-3-carboxy-4-nitroanilide   6 mmol/l R1               ______________________________________                                    

3.2.2. γ-GT reagent with peroxide additive:

Like 3.2.1. except that H₂ O₂ is additionally added to R1 at aconcentration of 147 mmol/l (0.5 ml 30% perhydrol per 30 ml R1) (H₂ O₂final concentration in the test=116 mmol/l).

3.3. Procedure for the γ-GT determination:

The γ-GT was determined on a Boehringer Mannheim/Hitachi 717 analyser at37° C. with the instrument settings according to the workinginstructions of the package insert (15 μl sample, 250 μl R1, 50 μl R2;wavelengths 660/405 nm; measurement interval 30th-50th measurementpoint).

3.4. Results

3.4.1. Reaction kinetics (absorbance time course):

FIG. 5 shows the absorbance time course in the γ-GT test when using thebasic reagent (3.2.1) and the following samples:

curve 1: 0.9% NaCl (=reagent blank value)

curve 2: serum without DClHb

curve 3: serum containing 400 mg DClHb/dl

curve 4: serum containing 800 mg DClHb/dl

curve 5: serum containing 1200 mg DClHb/dl

curve 6: serum containing 1600 mg DClHb/dl

curve 7: serum containing 2000 mg DClHb/dl

FIG. 5 shows that a base absorbance of more than 2 is already reached inthe conventional γ-GT test at a DClHb concentration of 800 mg/dl whereasthe measuring range of the photometer is exceeded by the samples spikedwith more than 1600 mg/dl DClHb and thus from the outset it is no longerpossible to determine γ-GT.

In contrast, as shown in FIG. 6 (assignment of the absorbance timecourses to the various samples as in FIG. 5) the presence of peroxide inR1 of the γ-GT reagent leads to a rapid decrease of the initialabsorbance caused by the haemoglobin derivative so that an initialabsorbance of less than 1 is reached at the start of the measurementinterval (measuring point 30) even with the serum spiked with DClHb to2000 mg/dl.

A further haemoglobin bleaching effect which may still underly thecolour signal from the conversion of the chromogenic substrate can alsoin this case be determined analogously to examples 1 and 2 in a paralleltest mixture with 1 containing H₂ O₂ and with R1 of the base reagent aschromogen-free R2 (curves 1'-7' of FIG. 7) and taken into account whencalculating the γ-GT recovery.

3.4.2. γ-GT recovery

The following table lists the data for the γ-GT recovery in the serumpool spiked with various DClHb amounts when using (a) the conventionalγ-GT reagent and (b) the γ-GT reagent to which H₂ O₂ was added to R1:

    ______________________________________                                                    Recovery of γ-GT*                                                   DClHb con-                                                                              Reagent without                                                                           Reagent with                                      Sample centration  H.sub.2 O.sub.2 addition H.sub.2 O.sub.2 addition**       No.    (mg/dl)   U/l    %      U/l    %                                      1       0         53.3   (100)  52.3   98                                       2        200      31.9     60                49.6      93                     3        400      17.0     32                48.2      90                     4        600  -1.7    -3               49.8      93                           5        800  -9.1   -17               48.5      91                           6       1000  -24.8   -47              48.1      90                           7       1200  -30.0   -56              49.4      93                           8       1400  -30.1   -56              51.9      97                           9       1600   - 9.5  -18              51.8      97                           10       1800  -10.4   -19              50.0      94                          11       2000  -10.3   -19              50.8      95                        ______________________________________                                         *) In each case the tests were calibrated with a calibrator for automated     systems, Boehringer Mannheim GmbH, Order No. 759 350                           **) corrected by subtracting the kinetic blank value test mixture with       chromogenfree R2)                                                        

The able shows that the recovery of γ-GT with the conventional reagentdecreases very rapidly with increasing DClHb concentrations and alreadyresults in negative values above 600 mg/dl, whereas with the reagentcontaining the peroxide addition according to the invention it isalways>90%.

Furthermore with regard to the results obtained with the heamoglobinderivative from the Somatogen Co., the use of sodium perborate insteadof H₂ O₂ and the optional addition of catalase to R2, the statementsmade for the AP determination apply analogously.

We claim:
 1. A method for the elimination or/and reduction ofinterferences which are caused by the presence of free haemoglobin inthe determination of an analyte in a sample by optical measurement,whereinone or several peroxidic compounds are added to the reagent usedto determine the analyte or to a part thereof.
 2. The method as claimedin claim 1, whereininorganic peroxidic compounds are used.
 3. The methodas claimed in claim 2, whereinthe peroxidic compounds are selected fromthe group consisting H₂ O₂ and perborates.
 4. The method as claimed inclaim 1, whereinthe optical measurement is carried out at at least onemeasurement wavelength at which haemoglobin has an absorption.
 5. Themethod as claimed in claim 4, whereinthe optical measurement is carriedout at measurement wavelengths of 380-450 nm or/and 520-590 nm.
 6. Themethod as claimed in claim 1, whereinan analyte is determined selectedfrom the group consisting of α-amylase, alkaline phosphatase andγ-glutamyl transferase.
 7. The method as claimed in claim 1, whereinthefinal concentration of peroxidic compounds in the test mixture is 1-500mmol/l with respect to the content of O₂ ²⁻.
 8. The method as claimed inclaim 7, whereinthe final concentration is 5-200 mmol/l.
 9. The methodas claimed in claim 1, whereinthe duration of action of the peroxidiccompounds on the sample before measurement is at least 1 minute.
 10. Themethod as claimed in claim 1, whereinthe analyte determination iscarried out as a multi-step test in which at least two partial reagentsare added at different times to the sample.
 11. The method as claimed inclaim 10, whereinthe peroxidic compounds are added together with thefirst partial reagent.
 12. The method as claimed in claim 1, whereinasample is determined which contains a blood substitute.
 13. The methodas claimed in claim 1, whereinthe determination is carried out on aserum or plasma sample.
 14. The method as claimed in claim 1, whereinthedetermination is carried out in an automated analyzer.
 15. The method asclaimed in claim 1, whereinthe measurement signal obtained in theanalyte determination is subjected to a blank value correction.
 16. Themethod as claimed in claim 15, whereinthe kinetic blank value issubtracted from the measurement signal.
 17. A reagent kit for thedetermination of an analyte in a sample by optical measurement,whereinin addition to the components required for the analytedetermination, it contains at least one peroxidic compound to eliminateor/and reduce interferences which are caused by free haemoglobin. 18.The reagent kit as claimed in claim 17, whereinit is composed of atleast two partial reagents that are spatially separated from one anotherin which one partial reagent contains the peroxidic compound and theother partial reagent or reagents contain additional test components.19. The reagent kit as claimed in claim 17 for the determination ofα-amylase preferably comprising a first partial reagent which containsthe peroxidic compound, a second partial reagent which contains asuitable buffer and optionally an α-amylase auxiliary enzyme or/and anantibody and a further partial reagent which contains a chromogenicα-amylase substrate.
 20. The reagent kit as claimed in claim 17 for thedetermination of alkaline phosphatase comprising a first partial reagentcontaining a peroxidic compound, a second partial reagent which containsa suitable buffer and a further partial reagent which contains achromogenic substrate for alkaline phosphatase.
 21. The reagent kit asclaimed in claim 17 for the determination of γ-glutamyl transferasecomprising a first partial reagent containing a peroxidic compound, asecond partial reagent which contains a suitable buffer and a furtherpartial reagent which contains a chromogenic substrate forγ-glutamyl-transferase.